Relevance to public health: Heterochronic genes control timing of cell differentiation and are related to human cancer genes, but little is known about their mechanism of action. In this proposal, we will identify genes that function with heterochronic genes to control timing of cell differentiation. We predict that these genes are likely to reveal novel development/cancer genes and mechanisms of development/cancer. Specific Aims: During animal development, tissues and organs form in a particular temporal sequence, but little is known about the mechanisms involved in temporal patterning. In C. elegans, the timing of epidermal seam cell development is regulated such that seam cells divide with a stem cell-like lineage during larval development and terminally differentiate at the adult stage, providing an excellent model for temporal control of cell fate, proliferation and differentiation. A pathway of heterochronic genes, which includes at least 2 microRNAs (miRNAs) and their targets, controls seam cell timing, but the exact mechanisms used by these genes are not understood. To shed light on the temporal patterning mechanisms employed in seam cells, we propose to use the power of C. elegans genetics to identify their targets and effectors, all potentially new heterochronic genes. Ultimately, this work will not only expand our understanding of the mechanisms and genes that contribute to temporal control in C. elegans, but given the homology of C. elegans heterochronic genes to human cancer genes, we believe that this work will be directly applicable to human development and disease, as well as timing in other less well-studied areas, such as aging. Aim 1: In previous work, we have shown that hbl-1 controls the timing of seam cell development, providing us with a foothold to understand developmental timing in this powerful model organ. To elucidate the mechanisms used by this transcription factor to control developmental timing, we propose to identify downstream effectors of hbl-1. We predict that these effector genes will be new heterochronic genes themselves, and may point to their human homologues as potential mammalian development and cancer genes. Aim 2: We propose to test if hbl-1 interacts with a second transcription factor, EGL-35 to control timing of seam cells. Aim 3: We have shown that aging is a timed event under control of heterochronic genes in C. elegans, but little is known about their mechanism for gene control in aging animals. We propose to determine how these age-regulating genes interact with the insulin-signaling pathway, identify their site of action and identify their downstream effector genes (possibly new developmental timing genes) to help elucidate this mechanism. This work may shed light on mechanisms of human aging.